A Closed-Cycle Capillary Polymerase Chain Reaction Machine

Anal. Chem. 2001, 73, 2018-2021

A Closed-Cycle Capillary Polymerase Chain Reaction Machine Jeff Chiou,† Paul Matsudaira,‡ Ain Sonin,§ and Daniel Ehrlich*

Whitehead Institute, 9 Cambridge Center, Cambridge, Massachusetts 02142

A novel thermocycling machine based on a microcapillary equipped with bidirectional pressure-driven flow and in situ optical position sensors is described. A 1-µL droplet of reaction mixture moves between three heat zones in a 1-mm-i.d., oil-filled capillary using a multielement scattered light detector and active feedback. Dwell times and accelerations can be adjusted independently. As a demonstration of the device, 30 cycles of a 500-base pair product were performed in 23 min with 78% amplification efficiency. This result compares well with previous highspeed thermocyclers. Theoretically, the arrangement can approach a time of 2.5 min for 30 cycle amplifications of a 500-base pair product. Polymerase chain reaction (PCR) is a molecular biology technique for exponentially increasing the number of copies of a specified portion of the initial template DNA. DNA can exist in either double-stranded (dsDNA) or single-stranded (ssDNA) forms. DNA length is specified by the number of its bases (ssDNA) or base pairs (dsDNA), and the efficacy of PCR amplification machines is specified in terms of the efficiency of sample doubling per three-step thermocycle (DNA denaturing, annealing, and extension) for a given DNA strand length in base pairs (bp). The most common definition is PCR efficiency Y. The end concentration of product DNA divided by the initial concentration of template DNA is (1+Y)n, with n being the number of cycles. Initial template concentration is such that PCR reactions typically consist of 20-45 cycles.1 PCR can result in product concentrations greater than 1 million times that of the initial template. However, it is important to note that amplification efficiency generally deteriorates with increasing strand length. A standard commercial PCR machine is a computer-controlled heater block holding tubes of PCR solution. Its reaction time is dominated by the temperature transitions, typically 2 °C/s, which limits 30 PCR cycles of a 500-bp product to a minimum processing time of 1-2 h. Theoretically, PCR can be performed much more quickly. It has been shown2 that annealing and denaturing can take place almost instantly. Therefore, PCR time is ultimately * To whom correspondence should be addressed. Tel: (617) 258-7283. Fax: (617) 258-7226. E-mail: [email protected]. † Tel: (617) 258-8110. Fax: (617) 258-7663. E-mail: [email protected]. ‡ Tel: (617) 258-5188. Fax: (617) 258-7226. E-mail: [email protected]. § Tel: (617) 253-2247. Fax: (617) 258 8559. E-mail: [email protected]. Massachusetts Institute of Technology. Room 3-256. 77 Massachusetts Avenue. Cambridge, MA, 02139. (1) Newton, C. R.; Graham, A. PCR; The Alden Press: Oxford, U.K., 1994. (2) Wittwer, C. T.; Garling, D. J. BioTechniques 1991, 10, 76-83.

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limited by extension time. According to manufacturer information (Promega; Madison, WI), Taq DNA polymerase can extend at 100 bases/s. Researchers have tried several methods to minimize PCR time. Wilding et al.3 and Woolley et al.4 both used a small silicon chamber instead of a heat block. The smaller thermal mass was intended to allow for more rapid temperature transitions and equilibrations. Wilding was not able to achieve results as efficient as a conventional cycler in comparable time (90 min). Woolley was able to amplify a 159-bp target from Salmonella genomic DNA over 35 cycles in ∼40 min. Wittwer and Garling2 put their aliquots in glass capillaries, which were cycled using heated and roomtemperature air. The low thermal mass of the air and capillaries, along with the high surface-to-volume ratio of the samples in the capillaries, allowed them to amplify a 536-bp product from predenatured (boiled) human genomic DNA in 40 min with yield similar to a conventional PCR machine. Nakano et al.5 did away with the need for heating and cooling a chamber altogether by pushing the liquid through a Teflon capillary strung 30 times through three different temperature zones. The high surface-tovolume ratio of the sample passing through the capillary permitted fast cycling. They obtained 1-kb product from 30 cycles but used high template concentration, so even for their longest PCR time (44 min), their efficiency was only 29%. However, they used Tth DNA polymerase, which is known to have a lower extension rate than Taq.6 Kopp et al.7 replaced the Teflon capillary with a microchannel lithographically etched in a glass wafer. Since the channel diameter was smaller, time was reduced even further. They amplified a 176-bp product over 20 cycles with ∼85% yield of a conventional PCR machine in 18.7 min. The speed of both Nakano and Kopp’s PCR machines can be varied by increasing the travel speed of the sample plug (Kopp reported product in as little as 1.5 min for 20 cycles), but efficiency drops as speed increases. The objective of this research was to produce a PCR machine to amplify a sample in as little time as possible with efficiency comparable to that of commercial PCR machines. An aliquot size (3) Wilding, P.; Shoffner, M. A.; Kricka, L. J. Clin. Chem. 1994, 40, 18151818. (4) Woolley, A. T.; Hadley, D.; Landre, P.; deMello, A. J.; Mathies, R. A.; Northrup, M. A. Anal. Chem. 1996, 68, 4081-4086. (5) Nakano, H.; Matsuda, K.; Yohda, M.; Nagamune, T.; Endo, I.; Yamane, T. Biosci., Biotechnol., Biochem. 1994, 58, 349-352. (6) Bej, A. K.; Mahbubani, M. H. In PCR Technology: Current Innovations; Griffin, H. G., Griffin, A. H., Eds.; CRC Press: Boca Raton, FL, 1994; pp 219-237. (7) Kopp, M. U.; de Mello, A. J.; Manz, A. Science 1998, 280, 1046-1048. 10.1021/ac001227f CCC: $20.00

© 2001 American Chemical Society Published on Web 03/22/2001

Figure 1. Apparatus schematic.

of 1 µL or smaller was chosen for potential compatibility with small-scale electrophoresis devices being developed in our laboratory.8 A further requirement was for the device to have independently adjustable denaturing, annealing, and extension conditions such as a commercial PCR machine. METHODS AND MATERIALS A schematic of our apparatus is presented in Figure 1. The PCR mix is in the form of a 1-µL drop, or “plug”, that moves inside of an otherwise oil-filled 1-mm-i.d. × 1/16-in.-o.d. PTFE capillary. The small aspect ratio of the sample plug (∼1.3 at rest) prevents it from breaking into pieces via the Rayleigh instability9sa potential problem given the fluid-in-fluid arrangement. Three heat blocks define the denaturing, annealing, and extension heat zones. A computer following a user-specified heating schedule moves the sample plug by controlling the pressures at the capillary ends and tracks its location using an optical detection system. This arrangement has several advantages. Like Nakano and Kopp’s devices, the sample plug moves through a small inner diameter heated capillary, so heating is fast. The travel time was 6 s/cycle at the pressures used in our experiments. Once the sample plug reaches a heat zone, it attains equilibrium temperature within ∼1.3 s (see Discussion). Since there are three heat steps, this is 10 s of heat transition per cycle, contrasted with more than 40 s/cycle for a conventional commercial PCR machine. Nakano and Kopp’s devices enjoy fast heating due to the same principles. However, in their devices, the sample plug is so long that it occupies multiple temperature zones simultaneously. Internal mixing can cause different DNA strands to see different time-temperature histories. By contrast, our arrangement utilizes a compact plug that is completely contained in a given zone. Nakano and Kopp move their sample plugs using syringe pumps that provide constant plug speed. While the speed can be adjusted to be faster or slower, the total number of cycles, as well as the ratios between the denaturing, annealing, and extension times, is fixed by the geometry in each device. Since our device uses closedloop computer-controlled intermittent reciprocating motion, it has neither constraint. Each heat block is a 1/2-in. cube machined out of aluminum. The high thermal conductivity of aluminum ensures good temperature uniformity within each block. The blocks are mounted on G-10 composite with 0.6-in. spacing between adjacent blocks to thermally insulate each zone. Each block is fitted with a (8) Schmalzing, D.; Tsao, N.; Koutny, L.; Chisholm, D.; Srivastava, A.; Adourian, A.; Linton, L.; McEwan, P.; Matsudaira, P.; Ehrlich, D Genome Res. 1999, 9, 853-858. (9) Rayleigh, L. Proc. London Math. Soc. 1878, 10, 4-13.

cartridge heater and thermocouple connected to a PID controller to maintain constant temperature. The capillary ends are attached via chromatography fittings to small oil reservoirs machined out of clear acrylic. Each reservoir is connected through computer-controlled solenoid valves to 5 psig, 7.5 psig, and atmospheric pressures. In addition, the righthand reservoir is connected to -1.5 and 1.5 psig sources in order to load the oil and sample plug. Once the sample plug is loaded into the capillary, the entire capillary can be pressurized to 5 psi to prevent sample degassing, which would interfere with the sensor system. This pressure value was figured as follows. The aliquot must go from refrigerated storage at 4 °C, 1 atm (0 psig) to 94 °C inside the apparatus. Using solubility data for air in water10 and Henry’s law (which states that gas solubility is proportional to the partial pressure of the gas above the solution), we calculated that air solubility at 94 °C, 3.1 psig is equivalent to that at 4 °C, 0 psig. A value of 5 psi provides a safety factor. In practice, it was found that the pressurization was necessary for 30-cycle runs but not for shorter 10-cycle runs. Pressure differentials across the reservoirs produce plug motion. A 2.5 psi pressure difference moves the plug at a maximum velocity of 1.4 cm/s. For 10 cycle runs, degassing was not a problem, so the (1.5 psig pressures at the right oil reservoir were used to move the plug, while the left reservoir remained at atmospheric pressure. For 30 cycles, the sample plug was moved by applying 7.5 psi to the reservoir the plug was to move away from and 5 psig to the other reservoir. Both reservoirs were pressurized to 5 psig when the plug was stationary. A He-Ne laser beam is focused onto the left entrance of the capillary. Since the index of refraction of mineral oil (n ) 1.48) is higher than that of PTFE (1.376), the beam waveguides down the oil in the capillary. It scatters upon reaching the curved oil/ plug interface. One photodiode is placed on either side of each block. The photodiodes capture the laser scatter, and their signals are amplified, filtered, and reported back to the computer. The scatter is detectable even when the sample plug is inside of an opaque heat block. Actual operation is as follows. The plug is loaded left of all blocks. The machine moves the plug to the right toward the first block. It checks the signal of the photodiode on the side of the block closest to the plug until it exceeds a certain threshold value. Then it checks to see when the difference between the signals of the photodiodes on both sides of the block drops below another threshold level. At this point, the plug is considered to be in the middle of the block. The pressure differential is set to zero, and timing of the heat step begins. The sample plug can drift inside the block, so if the difference in the block’s two photodiode signals exceeds a dead-band value, the machine pushes the slug back to the middle of the block. Once the particular heat step time is completed, the routine is repeated to execute the next heating step. RESULTS As a demonstration, we used our machine to perform a 10cycle PCR temperature optimization. Results are presented in (10) Dean, J. A. Lange’s Handbook of Chemistry; McGraw-Hill Book Co.: New York, 1985.

Analytical Chemistry, Vol. 73, No. 9, May 1, 2001

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Figure 3. Dependence of minimum heating, travel, and extension times on capillary inner diameter. Extension time is for a 500 bp product.

Figure 2. Typical 10-cycle temperature optimization. Solid line is average of data points (n ) 3). (a) Denature temperature optimization. TD ) 88, 90, 92, 94, 96, and 98 °C; TA ) 70 °C; TE ) 72 °C. (b) Anneal temperature optimization. TD ) 94 °C; TA ) 54, 56, 60, 64, 68, 70, and 72 °C; TE ) 72 °C. (c) Extension temperature optimization. TD ) 94 °C; TA ) 70 °C; TE ) 70, 72, 74, 76, 78, and 80 °C.

Figure 2. The PCR mix consisted of ∼0.18 units/µL Taq, 1.9 pM λ phage template DNA, 17 nM 524 base pair product, 600 nM each primer, 300 µM each dNTP base, 10 mM Tris-HCl (pH 9.0), 2 mM MgCl2, and 50 mM KCl. Product was included in the initial mix to compensate for the low number of cycles. Biological detergent Triton X-100 was extracted from the purchased Taq via centrifugal concentration to ensure high surface tension. This step introduces some ambiguity in Taq activity, but was required for our trials, since we could not find a source of detergent-free Taq. One mix was used for each optimization; variability of mixes and the nature of the PCR reaction accounts for yield variability. Each cycle consisted of TD, 2 s (5 s for cycle 1) f TA, 2 s f TE, 30 s. TD, TA, and TE are denaturing, annealing, and extension temperatures, respectively. The default TE and TA were almost the same 2020 Analytical Chemistry, Vol. 73, No. 9, May 1, 2001

(72 and 70 °C, respectively) and were determined by rough optimization in a conventional PCR machine with slow-temperature transitions. Yields were quantified on a 1% agarose gel stained with SYBR Green I nucleic acid stain. Results were qualitatively as expected. In Figure 2a, higher temperatures result in more complete denaturing and hence greater yield. In Figure 2b, The 56 °C optimal annealing temperature corresponds with primer manufacturer data. In Figure 2c, the extension temperature is generally consistent with the reported optimal temperature range of ref 6, 70-80 °C, although the optimum may prove to be at lower temperature due to the removal of the Triton X-100. The authors did not test TE < 70 °C, though this range is definitely of interest for any further experiments. To characterize the system under high-cycle number conditions, nearly 200 successful 30-cycle runs have been completed. Typical run times are 23 min for 30 cycles, using conservative dwell times. Typical efficiency is Y ) 78% for 47 s/cycle, 500-bp product. This time compares favorably with a commercial PCR machine (typically Y ) 70%, 3 min/cycle, 500-bp product) and Kopp’s arrangement7 (Y ≈ 70%, 56 s/cycle, 176-bp product) at comparable efficiencies using Taq. DISCUSSION This novel PCR machine combines the efficient heating of the fastest experimental machines with the programmable cycling flexibility of a commercial machine. Its small aliquot volume, 1 µL, could allow substantial savings in expensive reagents. Future work on this design can be directed toward automation of loading, multiplexing, and even faster cycle times. Faster PCR times can be achieved by scaling down our system. Figure 3 shows how components of minimum cycle time vary with apparatus size for a 500 bp product. It is assumed that the block size and distance between blocks scale with tube diameter d. One component of cycle time is the time required to heat and cool the sample plug. This was conservatively estimated by modeling the sample plug as a cylinder initially at uniform temperature in which heat transfer takes place solely by radial conduction. The product of density and specific heat for Teflon

(1.25 × 105 kJ/(m3 K) at a median block temperature of 70 °C) is much higher than that of mineral oil (1.83 × 103 kJ/(m3 K) at 70 °C), so the capillary wall temperature is modeled as constant. The greatest temperature transition (other than the room temperature to initial denature transition, which is discounted since the initial denature time is much longer than subsequent denature steps) is between annealing and denaturing, typically 50 and 94 °C, respectively. PCR samples are considered to be at the correct temperature if they are within 0.5 °C of the specified denaturing, annealing, or extension temperature. Therefore, the heating time was taken as the time for the cylinder, starting at a uniform temperature of 50 °C, to reach 93.5 °C along its centerline when its outer radius is exposed to a constant temperature of 94 °C. From conduction charts,11 it is determined for these conditions that Rt/r2 ≈ 0.85, where R is thermal diffusivity, t is time, and r is cylinder radius (i.e., inner radius of the tube). The thermal diffusivity of oil (7 × 10-8 m2/s; see ref 12) is smaller than that of water (0.16 × 10-6 m2/s) in the temperature range of the blocks, so it is used to determine the worst-case heating time (even though the sample is an aqueous solution, it is flanked by mineral oil). Since there are three heat steps per cycle, this heating time is (9 s/mm2)d2, where d is the tube inner diameter. Another factor to consider is the minimum transit time. If the sample plug moves too fast, it will break into pieces. According to Olbricht and Kung,13 breakup is determined by rdrop/r, µdrop/µ, Fdrop/F, the Reynolds number Re ) 2vmaxrF/µ, and the capillary number Ca ) µv/σ. rdrop is the radius of the drop if it were a sphere, r is the tube inner radius, µdrop is the viscosity of the sample plug (taken to be equal to that of water), µ is the viscosity the oil, Fdrop is the density of the sample plug (taken to be equal to that of water), F is the density of the oil, v is the average fluid velocity, and σ is the surface tension at the oil/sample plug interface. For our machine, rdrop/r, µdrop/µ, and Fdrop/F are fixed by the plug geometry and liquids. vmax, the maximum v prior to breakup, was experimentally found to be ∼0.2 m/s, corresponding to a Ca of 0.26. For similar values of rdrop/r, µdrop/µ, and Fdrop/F, Olbricht and Kung found breakup at Ca ) 3.5, for Re < 0.1. However, in our case, Re ) 3.7. Re ∝ r, so decreasing r should allow the critical Ca to approach 3.5. As a conservative estimate, we assumed Ca ) 0.26 for all r, so vmax ) 0.2 m/s. The sample (11) Kreith, F.; Bohn, M. S. Principles of Heat Transfer; Harper and Row: Publishers: New York, 1986. (12) Klaus, E. E.; Tewksbury, E. J. In CRC Handbook of Lubrication (Theory and Practice of Tribology); Booser, E. R. Ed.; CRC Press: Boca Raton, FL, 1983; Vol. 2. (13) Olbricht, W. L.; Kung, D. M. Phys. Fluids A: Fluid Dyn. 1992, 4, 13471354. (14) Constantinescu, V. N. Laminar Viscous Flow; Springer-Verlag: New York, 1995. (15) Cha, R. S.; Thilly, W. G. In PCR Primer: A Laboratory Manual; Dieffenbach, C. W., Ed.; Cold Spring Harbor Laboratory Press: Plainville, NY, 1995; pp 37-51.

plug travels 4 in./cycle. Neglecting actuator dynamics, and noting that the fluid can reach steady-state speed14 within ∼Fr2/µ ≈ 3 ms, the travel time is ∼4 in./vmax ) 0.5 s for our system. Of course, this is at greater pressure differentials than used for the 10- and 30-cycle experiments. As the machine is scaled down, the travel distance scales with the diameter, so travel time ) (4in. × d)/ (1 mm × vmax) ) (0.5 s/mm)d. For small d, PCR time is ultimately dominated by the extension time, which does not scale with size. Taq can process DNA at 100 bases/s at 74 °C, so a 500-base DNA takes 5.0 s to extend. The diameter is limited by the total number of copies of template. Less than ∼100 template molecules will result in unreliable PCR. Concentrations of >5 × 10-14 M also hurt the reaction.15 Combining these two constraints and keeping the same sample plug aspect ratio, it is determined that the minimum capillary diameter is 0.11 mm. Cycle time is composed of the following factors: denaturing time, annealing time, extension time, travel time, and heating time. Annealing and denaturing have been shown to take place almost instantly.2 With good actuators, plug acceleration time is overshadowed by constant-velocity travel time. Figure 3 shows how the minimum times of the remaining significant factors scale with apparatus size. We can see that, for a 500-bp target, cycle time is completely dominated by extension time below d ) 0.3 mm. From this perspective, it is not worth scaling down to smaller diameters. Of course, the point of diminishing returns varies with product length. In summary, we have constructed and tested a novel capillary PCR machine. Ten-cycle PCR optimization results have been presented to demonstrate its viability. Thirty-cycle PCR runs have been conducted which result in up to 78% efficiency for 30-cycle PCR of a 500-bp target from genomic λ-DNA in 23 min. This compares well with conventional PCR machines that take 1-2 h to perform the same task, as well as machines in the literature for comparable efficiency. Times can be improved by scaling down the device, but even in the theoretical case, reducing the diameter below 0.3 mm does not help when trying to amplify a product 500 base pairs or longer. Based on the arguments presented, it is estimated that a minimum geometry machine using our design could achieve PCR of a 500-bp product in 5.2 s/cycle. ACKNOWLEDGMENT This work was supported by the National Institutes of Health (grant HG01389) and the Air Force Office of Scientific Research (grant F49620-98-1-0235). Received for review October 17, 2000. Accepted February 17, 2001. AC001227F

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